The central objective of this research is to define the ways in which drugs interfere with cholinergic systems on subcellular, cellular and system levels. Methods have been devised to estimate both levels and turnover of acetylcholine and choline using gas chromatography/mass spectrometry in conjunction with deuterium-labelled internal standards and tracers. These methods will be improved and extended by development of multiple labelling techniques, separate estimation of rates of synthesis and degradation of acetylcholine, and regional turnover measurements in discrete brain areas and in subcellular organelles. Rates of release of choline and acetylcholine from cerebral cortex, ventricles and push-pull cannulae will be measured both with and without topical anticholinesterase treatment. The influence of electrolytic lesions at remote sites on turnover and release will also be studied. The methods will be used to define the effects of drugs on cholinergic systems, the anatomical and biochemical pathways mediating these effects and their relation to the behavioral and pharmacological actions of the drugs. Compounds to be studied include anticholinergic agents (atropine, scopolamine, Ditran), antidepressants (amitriptyline, DMI), neuroleptics (chlorpromazine, haloperidol) cholinesterase inhibitors (physostigmine analogs, DFP), morphine, ethanol, amphetamine and 1-DOPA. Adaptive changes in cholinergic systems will be sought as possible explanations for tolerance to morphine, ethanol and DFP.